Huntington's disease (HD) belongs to a family of eight inherited, unbeatable neurodegenerative diseases caused by the abnormal expansion of a polyglutamine (polyQ) stretch. Many of these diseases are characterized by protein deposits called inclusion bodies (IBs), there pathogenic roles are controversial. We made a model of Huntington's disease with neurons that recapitulates polyQ-dependent neuron-specific death and IB formation. In the previous funding period, we built a robotic microscope to monitor pathological changes in single neurons over time and to quantitate the extent to which a specific early change predicts whether a neuron lives or dies. We found that the risk of death in neurons with polyQ-expanded huntingtin (htt) decreases, at least temporarily, upon IB formation but it increases with polyQ length in neurons without IBs. We developed an antibody (mAb) 3B5H10 that selectively binds a disease-associated conformation of polyQ in htt, which is reduced upon incorporation of htt into an IB. We hypothesize that IB formation is a beneficial coping response by neurons to sequester more toxic, soluble forms of polyQ-expanded htt. Recognition of htt by mAb 3B5H10 predicts neuronal death, suggesting that this polyQ conformation might be pathogenic. Levels of polyQ-expanded htt increase before IB formation, possibly indicating a defect in htt degradation. However, it is not known whether impaired proteasome degradation of htt causes IB formation or leads to polyQ-dependent death. Whether IB formation is protective per se or simply a marker of longevity is also unclear. The structure of a disease-associated polyQ stretch is not known, nor is it clear whether aggregation intermediates of polyQ-expanded htt predict death better than the conformation bound by mAb 3B5H10. We will use the tools developed during the previous period to accomplish the following Specific Aims: Specific Aim 1. To assess the relationship between proteasome function and IB formation and determine if proteasome dysfunction is a major predictor of polyQ-dependent death. Specific Aim 2. To determine if IB formation provides a measure of protection against the deleterious effects of polyQ-expanded htt on neuronal function and survival. Specific Aim 3. To elucidate neurotoxic species of polyQ-expanded htt.